DESCRIPTION: (applicant's abstract) The overall goal of this proposal is to study the responses of cells to high concentrations of heavy metals. Certain metal ions, including those of copper and zinc, are essential for a variety of metabolic functions of the cell, and these ions have to be efficiently scavenged from the environment. On the other hand, organisms can be exposed to high concentrations of heavy metals ions through metabolic disorders, polluted environments, or metal contaminated food. Thus, cells have to protect themselves against the damage caused by toxic concentrations of heavy metal ions. Here, reporter gene fusion technology is employed to study metal dependent gene expression in a prokaryotic model system. A random mutagenesis protocol is used to generate a mutant bank of the bacterium Pseudomonas fluorescens with the lac Z-based reporter gene transposon Tn5-B20. By monitoring the lacZ encoded beta-galactosidase activity, genes responding to the presence of heavy metal ions will be identified and their expression in response to heavy metals analyzed. The hypothesis to be tested is that cells will respond to the exposure of toxic heavy metals, such as cadmium, with the expression of a specific set of genes. Some of these genes will be involved in the specific protection mechanism of the cell against metal ions, others will be part of the general stress response of the cell. The reaction of the cell to toxic cadmium ions will be compared with the response of the cell to the essential metal ions of copper and zinc.